Lethal Interaction of Nuclear and Mitochondrial Genotypes in Drosophila melanogaster

! 1! Lethal interaction of nuclear and mitochondrial genotypes in Drosophila melanogaster Tiina S. Salminen*,1, Giuseppe Cannino*,1, Marcos T. Oliveira†, Päivi Lillsunde*, Howard T. Jacobs*,‡,2, and Laurie S. Kaguni*,§,2 *Faculty of Medicine and Health Technology, FI-331014, University of Tampere, Finland †Departamento de Tecnologia, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista "Júlio de Mesquita Filho", Jaboticabal, SP 14884-900, Brazil ‡Institute of Biotechnology, FI-00014, University of Helsinki, Helsinki, Finland §Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824, USA 1 The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 2 Both HTJ ([email protected]) and LSK [email protected]) are corresponding authors. G3: Genes|Genomes|Genetics Early Online, published on May 10, 2019 as doi:10.1534/g3.119.400315 © The Author(s) 2013. Published by the Genetics Society of America. ! 2! Running title Nuclear-mitochondrial lethality in Drosophila Keywords cybrid, respiration, cytochrome b, mtDNA copy number, melanotic nodules Correspondence Laurie S. Kaguni, Department of Biochemistry and Molecular Biology, 603 Wilson Rd., Michigan State University, East Lansing, MI 48824 ! 3! ABSTRACT Drosophila melanogaster, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko25t mutation, affecting mitochondrial protein synthesis. When combined with tko25t, the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko25t but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko25t mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease. ! 4! INTRODUCTION In virtually all eukaryotes, mitochondrial functions are maintained by a combination of genes located in two physically and functionally separate compartments: the nucleus and the mitochondrion. Mitochondrial DNA (mtDNA), typically present in many copies and inherited uniparentally, encodes only a small subset of the polypeptides required for the enzymatic functions of oxidative phosphorylation (OXPHOS). Because of maternal inheritance and the consequent lack of sexual recombination, combined with rapid segregation to homoplasmy in the germline, the mtDNA of a given individual may be considered as a haplotype. In addition to 13 polypeptide subunits of OXPHOS complexes I, III, IV and V (cI, cIII, cIV, cV), involved in stepwise respiratory electron flow and ATP production, metazoan mtDNAs encode the RNA components (2 rRNAs and 22 tRNAs) of the separate translation system inside mitochondria. The remaining polypeptides of the OXPHOS complexes, as well as all others involved in mitochondrial metabolism, are nuclear-coded, and must be imported and assembled correctly inside the organelle. Because these functions are indispensible for life, mutations in either nuclear or mtDNA that impair OXPHOS or related processes, can result in drastic phenotypic consequences up to lethality. Examples may be found from the plant, fungal, protist and animal kingdoms. Nuclear and mtDNA may be considered to be a coevolving system (Sloan 2015), with taxon-specific features themselves representing evolutionary adaptations to the environment (Otten and Smeets 2015). In humans, OXPHOS mutations in both genomes have been widely studied because they are recognized as important genetic causes of disease. Because of the huge range of pathological phenotypes that characterize mitochondrial disorders, both within and between families, combined with the lack of a reliable model system, unraveling the precise contributions of nuclear and mitochondrial genotype to OXPHOS disease remains problematic. For example, one of the most common mtDNA-determined diseases, Leber's Hereditary Optic Neuropathy (LHON) shows approximately 5-fold greater penetrance in men than women. The attractive idea that this may be due to one or more X-chromosomal modifiers is broadly supported ! 5! (Hudson et al. 2005; Shankar et al. 2008), although the precise loci involved have not been identified, and some studies have proven inconclusive with respect to specific populations, e.g. Ji et al. 2010). Similarly, the role of mitochondrial haplotype as a phenotypic risk-factor or modifier of many pathological traits, such as neurodegeneration (Keogh and Chinnery 2015), has been extensively considered, but remains controversial. In most such instances, conclusions rest on statistical arguments regarding the size and nature of cohorts and the representativeness of population-based sampling. In principle, because of strict maternal inheritance of mtDNA in most metazoans, the effects of different combinations of nuclear and mtDNA genomes can be studied experimentally by a back-crossing approach to create cybrid strains in which a given mtDNA haplotype is introgressed into different nuclear backgrounds. Drosophila melanogaster is a suitable organism for such studies because of its relatively short generation time (egg to sexually mature adult in ~12 d at 25 °C) and the plethora of available, well-characterized strains and other genetic tools. In two previous studies using this approach (Chen et al. 2012, Salminen et al. 2017), we detected subtle modifier effects upon organismal phenotype, of different mtDNAs present in wild populations of D. melanogaster. In a controlled, wild-type nuclear background (Oregon R, maintained long-term in the laboratory in Tampere, Finland, which we designate ORT), we found systematic effects of mtDNA on the time taken to complete development and on OXPHOS activities. These were found to correlate with mtDNA copy number (Salminen et al. 2017). In particular, long developmental time was associated with elevated copy number, suggesting a fitness cost of specific mtDNA haplotypes that could be compensated partially by increased mtDNA content. In the other study (Chen et al. 2012), we introgressed different mtDNAs into a nuclear background bearing the tko25t mis-sense mutation (Shah et al. 1997) in the nuclear gene encoding mitoribosomal protein S12 (Royden et al. 1987). tko25t flies have significantly decreased levels and activities of all four OXPHOS complexes containing mitochondrial translation products (Toivonen et al. 2001), and exhibit a complex but mild organismal ! 6! phenotype of developmental delay, mechanical stress-induced seizures, sensitivity to antibiotics and to high-sugar diet, and impaired hearing and courtship (Toivonen et al. 2001, Kemppainen et al. 2016). Four mtDNA backgrounds were found to confer a partial suppression of the tko25t phenotype (Chen et al. 2012). In order to test for more dramatic manifestations of nuclear-mitochondrial interactions using this approach, we introgressed into the tko25t nuclear (ntko25t) background a set of mtDNAs from strains that were previously found to represent extremes of the spectrum of mtDNA copy number and developmental timing, when studied in the nuclear ORT (nORT) background (Salminen et al. 2017). The resulting cybrid strains revealed a clear-cut example of synthetic lethality between ntko25t and the mtDNA of strain KSA2 (mtKSA2), the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species. This provides a paradigm for understanding genetic interactions between nuclear and mtDNA relevant to other contexts, including human health and disease. ! 7! MATERIALS AND METHODS Drosophila melanogaster strains and culture D. melanogaster strains ORT, KSA2, WT5A, VAG1 (Salminen et al. 2017), and the tko25t mutant (Judd et al. 1972) were originally obtained from stock centers and maintained longterm on standard high-sugar diet (Kemppainen et al. 2016). Cybrid strains were derived as previously (Salminen et al. 2017) by repeatedly back-crossing females of a given strain to ORT or tko25t males. Cybrid strains used in the experiments are designated nX mtY, where X is the specified nuclear and Y the specified mtDNA background. In controlled crosses to measure pupal stage phenotypes (mtDNA copy number, respiration and supercomplex activities), and adult stage phenotypes (eclosion timing and life span), tko25t cybrids were balanced over FM7, containing the Bar-eye marker. To distinguish tko25t from balancer larvae, the balancer also contained the dominant Tb marker (Bloomington line 36849, genotype FM7a, P{w[+mC]=Tb[1]}FM7-A). Egg-to-adult development and lifespan assay Batches of 12 females and 6 males were mated and tipped 4 times at 24 h intervals, giving approximately 100-150 eggs per vial. Flies were reared at 25 °C on a 12 h light/dark cycle and 60% humidity, and eclosion was scored daily. To evaluate the effects of antimycin A treatment, flies were cultured on standard medium containing the sub-lethal dose of 5 µg/ml of the drug (Frei et al. 2005). Lifespan was measured using batches of 200 females and males transferred to fresh culture bottles every 2-3 d, with the number of dead flies recorded each time. Larval dissection Wandering stage L3 larvae were dissected in a droplet of PBS under light microscopy, using thin forceps. Freely floating melanotic nodules were counted for each larva. Hemolymph was diluted into a total volume of 100 µl of PBS and hemocytes were counted using an Accuri C6 flow cytometer (BD Biosciences), with forwardand side-scatter gating used to distinguish total hemocyte count from debris (Anderl et al. 2016). ! 8! Relative mtDNA copy number analysis Total DNA was extracted from pools of 10 late-stage pupae of each sex, in four biological replicates, and copy number relative to a nuclear DNA locus was determined by quantitative PCR as described previously (Salminen et al. 2017). Briefly, mtDNA was quantified using primers against the 16S rRNA gene, and nuclear DNA using primers against the RpL32 gene, based on calibration curves generated in parallel. Respiratory enzyme activities Respirometry and blue-native electrophoresis (BNE) with in-gel histochemistry were performed as previously (Salminen et al. 2017), using homogenates from pupae collected soon after pupariation. A set of mtKSA2 ntko25t pupal cases from this time point were dissected prior to the experiment, to verify that each contained a living individual, i.e. to be sure that the assay was being conducted prior to the onset of lethality. Briefly, respirometry used a Clark-type electrode, with successive additions of standard substrates and inhibitors for the different respiratory chain complexes. BNE used the NativePAGE Novex Bis-Tris gel system (Invitrogen Life Technologies), with approximately equal loading checked by running aliquots of the samples on an SDS-PAGE gel that was then stained with Coomassie blue, prior to BNE. In-gel histochemistry was performed separately for cI and cIV, using standard staining procedures. Note that the blue dye used in BNE interferes with the in-gel assay for cIII, for which reason cIII was separately assayed enzymatically, using the same homogenates, as follows. Reduced decylubiquinone was prepared by adding a large excess of dithionite to a decylubiquinone solution (25 mg dissolved in 2.5 ml dimethyl sulfoxide plus 400 µl water, acidified by the addition of 50 µl 0.1 N HCl). The initially deep-orange solution was magnetically stirred for >30 min until it became transparent. After a full reduction (> 4 h) and the sedimentation of excess dithionite, the colorless upper phase was centrifuged (10,000 gmax, 5 min) and the supernatant used in the assay. Ten µg aliquots of fly homogenates were added to a reaction mix containing 20 mM phosphate buffer, 100 µM cytochrome c, 20 µM KCN, 100 µM EDTA and 0.02% TWEEN® 20 (pH 7.5), with and without the addition of antimycin A to 1 µM. After 3 min of incubation, 5 µl of reduced decylubiquinol was added and, after mixing, absorbance at 550 nm was followed at 21 °C for 2 min, at 10 s intervals. ! 9! Complex III activity was inferred by subtracting the rate of absorbance change in the presence of Antimycin A from that in its absence. Molecular modeling The structural effects of amino acid polymorphisms in Drosophila cytochrome b (cyt b) were evaluated by mapping onto the crystal structure of Bos taurus cIII, PDB 1BGY (Iwata et al. 1998), using the Pymol mutagenesis tool (www.pymol.org) under default settings. Pymol was also used to analyze all structural details and to produce the figures. DNA sequencing Using the primers indicated in Table S1, the Drosophila UQCR-C1 (CG3731) gene was amplified in two fragments by long PCR, using the same DNA preparations as for mtDNA copy number analysis, and then cycle-sequenced. Statistical analysis mtDNA copy number variation and eclosion frequencies were analyzed by one-way ANOVA, combined with the post hoc Tukey HSD test (astatsa.com). Student’s t-test was used for pairwise comparisons of other data. Life-span data were analyzed using the Kaplan-Meier test (GraphPad Prism v7.00). Data Availability Strains are available on request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. ! 16! respiratory deficiency under stress conditions may activate a sensor mechanism to generate such a response. This is consistent with the elevated mtDNA copy number seen in the ntko25t cybrid strains studied here. Moreover, it was highest in ntko25t mtKSA2 pupae (Fig. 1B), which also showed a clear respiratory defect (Figs. 4, 5). Because the defect is associated with functionally significant coding-region mutations in KSA2 mtDNA, elevated copy number is most likely a consequence of respiratory stress rather than its cause, but any compensatory effect of copy number increase was insufficient to overcome lethality. Paradoxically, mtKSA2 was observed previously to manifest a low mtDNA copy number in wild-type nuclear backgrounds, despite manifesting relatively slow development (Salminen et al. 2017). Significance of melanotic nodules in mtKSA2 larvae Oxidative stress in the posterior signaling centre of the lymph gland is known to potentiate cytokine signaling that leads to overproduction of lamellocytes (Sinenko et al. 2012), the blood cells responsible for encapsulating and killing parasitic wasps (Rizki and Rizki 1992). Even in the absence of wasp infection, this can manifest as melanotic nodules (Sinenko et al. 2012), as also seen in mtKSA2 ntko25t, or in wild-type flies cultured in antimycin A medium (Table 1). The observed increase in total hemocyte count (Fig. 2B) is part of the same response (Hanratty and Dearolf 1993, Qiu et al. 1998). Although the response can be elicited by other means, such as aberrant Toll (Hanratty and Dearolf 1993) or JAK/STAT signaling (Qiu et al. 1998, Harrison et al. 1995), by chromatin alterations (Braun et al. 1998, Walker et al. 2011), or even just by cuticular wounding (Markus et al. 2005), the observed blood cell phenotype in mtKSA2 flies is also consistent with a primary respiratory chain defect, such as achieved elsewhere by knockdown of cI or cIV subunits (Sinenko et al. 2012). Spread of a deleterious mtDNA mutation mtKSA2 flies harbor a potentially deleterious mtDNA genotype, conferring catastrophic consequences in specific nuclear backgrounds or when exposed environmentally to OXPHOS poisons. Thus, its presence in the KSA2 strain requires explanation. First, in the nuclear background in which it arose (Salminen et al. 2017), as well as in other 'wild-type' nuclear backgrounds such as nORT (Salminen et al. 2017 and Fig. 1), mtKSA2 is apparently neutral with respect to developmental timing and other traits, including cIII activity. Thus, its ! 17! presence could simply be accounted for as genetic drift. Second, although lethal in the presence of high doses of antimycin A (which would be encountered only rarely in nature), the potentiation of lamellocyte differentiation in response to milder stress, as represented in hemocyte proliferation and the propensity to form melanotic tumors, is consistent with a status primed to resist parasitic infection (Eslin and Prévost1998). Fly strains are already known to vary with respect to susceptibility to such infection (Kraaijeveld and Godfray 1999, Gerritsma et al. 2013), and we speculate that mtDNA genotype could be a factor that predisposes to resistance. Parasite resistance seems particularly important in certain environments (Kraaijeveld and Godfray 1999), and may outweigh any negative effects of decreased OXPHOS efficiency, especially if nutritional resources are plentiful. Positive selection is less of a consideration for ntko25t, which arose as a result of a mutagenic screen (Judd et al. 1972), and has not been seen in wild populations. Moreover, it is associated with a courtship defect (Toivonen et al. 2001). However, the net effect of the latter on survival in the wild is not clear, because the mutation appears simultaneously to decrease male attractiveness and female discrimination (Toivonen et al. 2001). Nevertheless, tko25t is also a phenotypically mild mutation. Because mtDNA mutations affecting the mitochondrial translation system appear largely to escape purifying selection (Stewart et al. 2008) it is possible that genetic variants with similar effects to tko25t could exist in natural populations. It has been argued elsewhere (Hill 2018) that mitonuclear selection could play a role in mate choice, and Drosophila may be a convenient organism in which to test this hypothesis. Relevance to human populations and disease Our previous observation of mtDNA haplotypes acting as partial suppressors of ntko25t (Chen et al. 2012), combined with the present findings of synthetic lethality with a different mtDNA background, offer a potential paradigm for understanding mitochondrial genetics more widely, including in humans. 'Mitochondrial genotype', regarding both nuclear and mtDNA, is not simply a case of good versus bad. As here, it can specifically affect one or more OXPHOS complexes, can influence ROS production, signaling, mitochondrial heat production, substrate utilization and even tissue-specific phenotypes linked to any of these. Mitochondrial disease is known to exhibit sharp threshold effects (Rossignol et al. 20113), ! 18! and for overt pathology to be precipitated by commonly experienced external stresses, such as viral infection (Schapira 2000, Parikh et al. 2013), which also impact mitochondrial function (Scholte 1988). Thus, nDNA/mtDNA combinations that are entirely viable in one environment may be pathological or even lethal in another. Although tko25t was a laboratorygenerated mutant, disease-causing mutations in mitoribosomal protein genes have been reported in humans (Saada et al. 2007, Lake et al. 2017, Borna et al. 2019), almost always as homozygotes or compound heterozygotes. It is likely that milder mutations affecting the mitoribosome are segregating naturally in the human population which, in combination with an equally mild mutant mtDNA equivalent to mtKSA2, which was isolated by chance from the wild, could cause a severe pathological phenotype. Our findings thus provide a concrete example of highly deleterious mitonuclear interactions, and argue that the issue needs to be taken into account in considering the possible effects of mtDNA as a pathological co-factor or modifier (Chou and Leu 2015), as well as the advisability of donor-recipient matching in mitochondrial replacement therapy (Reinhardt et al. 2013, Wolf et al. 2015). Naturally arising mitonuclear incompatibilities have recently been proposed as a driver of selection during human evolution (Sharbrough et al. 2017). However, the complex genetics inherent to the ntko25t mtKSA2 combination implies that similar examples in the human population may be hard to recognize, and may have been overlooked, especially given the profusion of rare mtDNA variants (Kim and Schuster 2013). ACKNOWLEDGEMENTS We thank Merja Jokela, Tea Tuomela, Troy Faithfull and Milja Luukkonen for technical assistance, and Eric Dufour for advice with statistics. Financial support was provided by Academy of Finland (grants 118654, 139587, 256615 and 272376), National Institutes of Health (GM45295), Fundação de Amparo à Pesquisa do Estado de São Paulo (grant 2014/02253-6) and the EU (Marie Curie International Incoming Fellowship GA328988). Author contributions are as follows: TSS and GC conducted most of the laboratory work and data analysis, assisted by PL, and participated in experimental design and planning. MTO conducted molecular modeling and was also involved in experimental design and planning. ! 19! LSK supervised the laboratory work. HTJ and LSK designed the project, and together with TSS, drafted the manuscript, to which all authors contributed. ! 20! LITERATURE CITED Anderl, I., L. Vesala, T. O. Ihalainen, L. M. Vanha-Aho, I. Andó, M. Rämet, and D. Hultmark, 2016 Transdifferentiation and proliferation in two distinct hemocyte lineages in Drosophila melanogaster larvae after wasp infection. PLoS Pathog. 12: e1005746. Barr, C. 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Salminen et al, Figure 3 p< 0.05 p< 0.01 ** * ** 40 30 20 10 0 mtORT mtWT5A mtVAG1 mtKSA2 * A eclosion (% of eggs laid) 10 8 6 4 2 0 B tko25t /tko25t tko25t / FM7 tko25t / Y FM7 / Y n = 940 eclosion (% of eggs laid) Salminen et al, Figure 4 nORT ntko25t *** *** ** ** ●●● ● *** *** ** *** ●● ●● CD ●● ●●● ●●● **** *** *** *** *** *** ●●● *** ●●● ●●● A B 0 30 20 10 KSA2ORT VAG1 WT5A KSA2ORT VAG1 WT5A 0 60 40 20 ●● ** *** *** *** *** *** *** *** E F 0 200 150 100 50 Oxygen consumption, nmol/ ml Complex I Complex III Complex IV nmol O2.min-1.mg protein-1 mtDNA – ORT KSA2 VAG1 WT5A ORT KSA2 VAG1 WT5A ns ns Salminen et al, Figure 5 A A B B C C males females nORT ntko25t Salminen et al, Figure 6 CBovine M-TNIRKSHPLMKIVNNAFIDLPAPSNISSWWNFGSLLGICLILQILTGLFLAMHYTSDTTTAFSS * * ** **** * ***** ***** ********* ****** ********* * ***** Human M-TPMRKTNPLMKLINHSFIDLPTPSNISAWWNFGSLLGACLILQITTGLFLAMHYSPDASTAFSS * * **** ** *** ***** ***************** ************* * ** * Drosophila MNKPLRNSHPLFKIANNALVDLPAPINISSWWNFGSLLGLCLIIQILTGLFLAMHYTADINLAFYS * *** ** ** * ** * *** ***** ** *** ** * **** * **** Yeast M--AFRKSNVYLSLVNSYIIDSPQPSSINYWWNMGSLLGLCLVIQIVTGIFMAMHYSSNIELAFSS Refseq RQILCYNRRIPLHELEQRIDAVSVGNVRDVAMKYIYDRCPAVAAVGPVENLPDYNRIRSSMYWLRV ORT RQILCYNRRIPLHELEQRIDAVSVGNVRDVAMKYIYDRCPAVAAVGPVENLPDYNRIRSSMYWLRV tko25t RQILCYNRRIPLHELEQRIDAVSVGNVRDVAMKYIYDRCPAVAAVGPVENLPDYNRIRSSMYWLRV AB D Bovine cIII Drosophila cIII model